Effects of Anticoagulants on Clarity of the Agglutination in Coombs Test

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Indian Journal of Pathology: Research and Practice Volume 9 Number 1 (Part II), January - April 2020 IJPRP DOI: http://dx.doi.org/10.21088/ijprp.2278.148X.9120.37

Original Research Article

Siddhartha Shanker Sinha1, Yogesh Kumar Yadav2, Sonal Saxena3, Pooja Jaiswal4, Juhi Chauhan5

1Associate Professor, 3Assistant Professor, 4Associate Professor, 5Senior Resident, Department of Pathology, Integral Institute of Medical Sciences and Research, Lucknow, Uttar Pradesh 226026, India. 2Associate Professor, Department of Pathology, Autonomous State Medical College, Ayodhya, Uttar Pradesh 224001, India.

Corresponding Author: Abstract Yogesh Kumar Yadav, Associate Professor, Department of Pathology, Autonomous State Context: Antiglobulin (Coombs) test is very important test in Medical College, Ayodhya, Uttar Pradesh the detection of incomplete (IgG) antibodies in the blood, either 224001, India. in free form (Indirect coombs test) or bound to the red blood cell E-mail: [email protected] (Direct coombs test). There is a possibility that anticoagulants like Received on 14.11.2019 Ethylenediaminetetraacetic acid (EDTA), Citrate, etc used for the Accepted on 29.01.2020 collection of blood sample, may effect the end result of the test. Aims: The present study is aimed at comparison of the end result (the clarity of agglutination) between the coombs test performed on plain blood and anticoagulated blood (EDTA and Citrate). Settings and Design: Blood samples collected from blood donors at the time of blood donation in plain and anticoagulated blood (EDTA and Citrate). Samples were subjected to coombs test at various dilutions and agglutination was observed under the microscope. Methods and Material: 6 ml. of blood samples will be collected from the 100 blood donors at the time of blood donation in and 2 ml each transferred into; EDTA bottle, Citrate bottle and Plain bottle. The sample thus collected was used for performing the Anti-globulin test. 10% red cell suspension was sensitized with Anti D IgG (as supplied by Tulip Diagnostics) at different dilutions (1/10, 1/20, 1/30, 1/40). The washed saline suspension of RBC’s is treated with Anti- human globulin serum and agglutination is observed under a microscope at 5X objective. Statistical analysis used: Friedman test. Results: The degree of agglutination obtained by using plain blood samples at various dilutions, tend to be higher compared to the same obtained by using citrate and EDTA blood samples. The citrate blood samples gives intermediate pattern between plain blood samples and EDTA blood samples. Conclusions: The use of plain blood was found to give better results compared to citrate and EDTA blood in performing the antiglobulin test. The chances of missing the detection of low titres of anti-D IgG is more with EDTA blood samples when compared to citrate blood samples and least with plain blood samples. Keywords: Antiglobulin; Coombs; Anticoagulants, Ethylenediaminetetraacetic acid (EDTA), Citrate

How to cite this article: Siddhartha Shanker Sinha, Yogesh Kumar Yadav, Sonal Saxena, et al. Effects of Anticoagulants on Clarity of the Agglutination in Coombs Test. Indian J Pathol Res Pract. 2020;9(1 Part II):241–247.

Introduction a) Undiluted: Take 100 L of Red Blood Cells suspension and add 200 L of undiluted Coombs test (also known as antiglobulin test or anti-D IgG. AGT) refers to two clinical blood tests used in b) 1/10 Dilution: Take 100 L of Red Blood immunohematology and immunology. The two Cells suspension and 200 L of 1/10 types of Coombs tests are the direct Coombs test diluted anti-D IgG. (also known as direct antiglobulin test or DAT), and c) 1/20 Dilution: Take 100 L of Red Blood the indirect Coombs test (also known as indirect Cells suspension and 200 L of 1/20 antiglobulin test or IAT). diluted anti-D IgG. The more commonly used test, the Direct Coombs d) 1/30 Dilution: Take 100 L of Red Blood test, is used to test for autoimmune hemolytic Cells suspension and 200 L of 1/30 anemia. diluted anti-D IgG. Antiglobulin (Coombs test) is very sensitive e) 1/40 Dilution: Take 100 L of Red Blood and is an important test in the detection of Cells suspension and 200 L of 1/40 incomplete (IgG) antibodies in the blood either in diluted anti-D IgG. free form or bound to RBC. There is possibility that anticoagulants like ethylene diamine tetra acetic D. Mix well and incubate for 45 minutes. acid (EDTA) and citrate, etc used in the collection of E. Wash the incubated preparation for 3 times blood samples may affect the end result of the test. in saline. The present study is aimed at the comparison of F. Decant the supernatant completely. Prepare the end result (clarity of agglutination) obtained on a 5% saline suspension. performing Coombs test using plain, EDTA and G. Add 100 L of Coombs (anti human globulin) citrate blood samples. reagent to 50 L of 5% saline suspension. Mix well and immediately centrifuge at 1000 rpm Materials and Methods for 10 seconds. H. The sediment is agitated and transferred to a Blood samples were collected from 100 blood glass slide. donors at the time of blood donation. I. The agglutination is observed under a microscope at 5X objective. Method of collection of data J. The microscopic pictures are divided into 6 6 ml. of blood samples are collected from the groups: blood donors at the time of bleeding and 2 ml. each transferred into; EDTA bottle, Citrate bottle a) 4+: Almost 100% of RBC’s are clumped and Plain bottle. Blood samples are collected with large single or only few clumps. from the blood donors at the time of bleeding at b) 3+: About 75% of RBC’s are clumped blood bank. The samples thus collected were used with several large clumps which are for performing the Anti-globulin test. The Anti- visible even macroscopically. globulin test was done as follows: c) 2+: About 50% of RBC’s are clumped. A. Centrifuge the blood sample and discard the The clumps are many and small even plasma/serum. visible macroscopically. a) 0.5 ml of the Red Blood Cells sediment d) 1+: About 25% of RBC’s are clumped. is mixed with 4 ml of normal saline by The clumps are tiny and numerous. vigorous shaking. Macroscopically just visible. b) Centrifuge at 2000 rpm for 5 minutes. e) +/-: Less then 25% of RBC’s are clumped. The supernatant is removed and the Tiny clumps visible microscopically sediment is resuspended in the saline. only. This procedure is repeated 3 times. f) -ve: No clumps, evenly distributed RBC’s, B. 5% suspension of washed Red Blood Cells No clumps visible microscopically. was prepared. C. The Red Blood Cells are sensitized with Inclusion criteria anti-D IgG as follows:- Donor selection as per drug control act. No prior

Indian Journal of Pathology: Research and Practice / Volume 9 Number 1 (Part II)/January - April 2020 Siddhartha Shanker Sinha, Yogesh Kumar Yadav, Sonal Saxena, et al. 243 investigation of donor expect haemoglobin. Sample plain blood samples), Fig. 2 (donor’s citrate blood wad collected only from the tubing of the blood samples) and Fig. 3 (donor’s EDTA blood samples). bag. The results obtained from donor’s blood samples are presented in Table 1 (plain blood), Table 2 Exclusion criteria (citrate clood) and Table 3 (EDTA blood). RH -ve blood samples and those blood samples From the Tables and Charts it is also observed which contains cold auto agglutinins (those clump that:- in bovine albumin). • The degree of agglutination is maximum in undiluted samples and decline as the dilution Results increases. • Degree of agglutination is more in plain The application of Coombs test was done on Donor’s blood samples compare to those of citrate Blood and the results obtained are presented in the blood samples and EDTA blood samples. form of charts and table. • Between citrate and EDTA blood samples The results are presented in the Fig. 1 (donor’s the degree of agglutination is comparatively

Table 1: Number and percentage of agglutination in plain blood samples

-ve +/- 1+ 2+ 3+ 4+ Donor’s No. of No. of No. of No. of No. of No. of Sample % % % % % samples samples samples samples samples samples Plain Blood 0 0 0 0 0 0 0 0 35 35.0 65 65 UD Plain Blood 0 0 0 0 0 0 36 36 64 64.0 0 0 1/10 Plain Blood 0 0 0 0 32 32 68 68 0 0 0 0 1/20 Plain Blood 0 0 33 33 60 60 7 7.0 0 0 0 0 1/30 Plain Blood 47 47 53 53 0 0 0 0 0 0 0 0 1/40 Abbreviations: UD (undiluted)

60 -ve 50 +/- 40 1+ 30 2+

20 3+

10 4+

0 Plain Blood Plain Blood Plain Blood Plain Blood Plain Blood UD 1/10. 1/20. 1/30. 1/40. Fig. 1: Percentage and results of donor’s blood samples in plain blood.

Indian Journal of Pathology: Research and Practice / Volume 9 Number 1 (Part II)/January - April 2020 244 Effects of Anticoagulants on Clarity of the Agglutination in Coombs Test

Table 2: Number and percentage of agglutination in citrate blood samples

-ve +/- 1+ 2+ 3+ 4+ Donor’s No. of No. of No. of No. of No. of No. of Sample % % % % % % samples samples samples samples samples samples Citrate UD 0 0 0 0 0 0 0 0 51 51 49 49 Citrate 1/1 0 0 0 0 6 6 50 50 44 44 0 0 Citrate 1/2 0 0 6 6 48 48 46 46 0 0 0 0 Citrate 1/30 6 6 50 50 44 44 0 0 0 0 0 0 Citrate 1/4 55 55 45 45 0 0 0 0 0 0 0 0 Abbreviations: UD (undiluted)

40 -ve

+/- 30 1+

20 2+

3+ 10 4+

0 Citrate Citrate Citrate Citrate Citrate UD 1/10. 1/20. 1/30. 1/40. Fig. 2: Percentage and results of donor’s blood samples in citrate blood.

Table 3: Number and percentage of agglutination in EDTA blood samples

-ve +/- 1+ 2+ 3+ 4+ Donor’s No. of No. of No. of No. of No. of No. of Sample % % % % % % samples samples samples samples samples samples EDTA UD 0 0 0 0 0 0 0 0 60 60 40 40 EDTA 1/10 0 0 0 0 0 0 65 65 35 35 0 0 EDTA 1/20 0 0 3 3 69 69 28 28 0 0 0 0 EDTA 1/30 24 24 53 53 23 23 0 0 0 0 0 0 EDTA 1/40 81 81 19 19 0 0 0 0 0 0 0 0 Abbreviations: EDTA (Ethylenediaminetetraacetic acid), UD (undiluted)

70 -ve 60 +/- 50 1+ 40 2+ 30 3+ 20 4+ 10

0 EDTA EDTA EDTA EDTA EDTA UD 1/10. 1/20. 1/30. 1/40. Fig. 3: Percentage and results of donor’s blood samples in Ethylenediaminetetraacetic acid blood

Indian Journal of Pathology: Research and Practice / Volume 9 Number 1 (Part II)/January - April 2020 Siddhartha Shanker Sinha, Yogesh Kumar Yadav, Sonal Saxena, et al. 245

more with citrate than EDTA blood samples. The details of 25th percentiles, 50th median and th Sixty- ve percent of donor’s blood sample 75 percentiles along with Friedman test value showed 4+ agglutination with undiluted samples and p value of donor’s blood samples at various when plain blood samples are used. This percentage dilutions of plain blood samples are presented in drops down to 49% with donor’s blood undiluted Table 4, of citrate blood samples in Table 5 and of samples when citrate blood samples are used; and EDTA blood samples in Table 6. 40% donor’s undiluted blood samples when EDTA From the Tables 4, 5 and 6 it is noted that blood samples are used. the degree of agglutination is maximum with Whereas when samples with maximum dilution undiluted blood samples and minimum or nil (1/40) are used negative results (no agglutination) with 1/40 dilutions and there is a study decline as are obtained with 47% donor’s blood samples when the dilutions increases which is evident from the plain blood sample is used. 55% of donor’s blood Friedman test value which is between 380 to 400 in samples when citrate blood samples is used. 81% all 3 types of samples of both categories. Similarly donor’s blood samples with EDTA blood samples. the p value is consistently less than 0.001 in all 3 samples which shows highly signi cant deference

Table 4: Donor’s blood samples of plain blood in various dilutions - Friedman test and P-value

Donor’s Sample N 25th Percentiles 50th (Median) 75th Percentiles Friedman test value p-value Plain Blood UD 100 4.00 5.00 5.00 392.353 p < 0.001 Plain Blood 1/10 100 3.00 4.00 4.00 HS Plain Blood 1/20 100 2.00 3.00 3.00 Plain Blood 1/30 100 1.00 2.00 2.00 Plain Blood 1/40 100 .00 1.00 1.00 Abbreviations: UD (undiluted)

Table 5: Donor’s blood samples of citrate blood in various dilutions-Friedman test and P-value

Donor’s Sample N 25th Percentiles 50th (Median) 75th Percentiles Friedman test value p-value Citrate UD 100 4.00 4.00 5.00 369.494 p<0.001 Citrate 1/10 100 3.00 3.00 4.00 HS Citrate 1/20 100 2.00 2.00 3.00 Citrate 1/30 100 1.00 1.00 2.00 Citrate 1/40 100 00 00 1.00 Abbreviations: UD (undiluted)

Table 6: Donor’s blood samples of EDTA blood in various dilutions-Friedman test and P-value

Donor’s Sample N 25th Percentiles 50th (Median) 75th Percentiles Friedman test value p-value

EDTA UD 100 4.00 4.00 5.00 387.893 p < 0.001 EDTA 1/10 100 3.00 3.00 4.00 EDTA 1/20 100 2.00 2.00 3.00 EDTA 1/30 100 1.00 1.00 1.00 EDTA 1/40 100 00 00 00 Abbreviations: EDTA (Ethylenediaminetetraacetic acid), UD (undiluted) in the end results of undiluted and various dilution as the Coombs’ test.1 This immunohematologic samples. technique has become a important tool in detecting immune-mediated hemolytic anemia, non immune Discussion hemolytic anemia, hemolysis of the newborn and transfusion reactions.1,2 The direct antiglobulin test (DAT) is a method of demonstrating the presence of The antiglobulin test was rst introduced by Dr immunoglobulin (Ig) and/or complement, bound Robin Coombs in 1945, since than this test is referred to the surface of red blood cells (RBCs) by the use

Indian Journal of Pathology: Research and Practice / Volume 9 Number 1 (Part II)/January - April 2020 246 Effects of Anticoagulants on Clarity of the Agglutination in Coombs Test of anti-human globulin to form a agglutination to go into the bag, then a small portion of this blood reaction which can be visibly detected. The test is squeezed into a glass tube for performing various is very speci c and sensitive in the detection of tests. The Red Blood Cells from the citrated blood erythrocytic auto and alloantibodies.3,4 Although sample is used for doing a Coombs cross-match an immune mechanism for hemolysis, the DAT and for the detection of irregular antibodies. In the may have certain pitfalls it can be negative for present study the blood samples were collected certain reasons. Autoantibodies which cause from the tubings directly in plain bottle, EDTA hemolysis perticularly IgA or IgM have a negative bottle and Citrate bottle (without prior mixing DAT because in this test we can only detect RBC ACD/CPD). that are coated with IgG or C3d.5 When lower titer The present study as revealed that of potent antibodies present, the DAT may fail to detect them.6 when adequate titer of low af nity 1. The degree of agglutination is maximum antibodies are present which are clinically relevant with undiluted blood samples and reduces may get removed at the time of washing in DAT as the dilution increases. This indicates the testing resulting in negative test.7 However, the degree of agglutination is proportionate to conventional method of Coomb’s test is stilll highly titre of anti-D IgG. effective and reliable, even after the development 2. The degree of agglutination obtained by using of newer methods like microtiter plates, gel plain blood samples at various dilutions, tend 8 microcolumns, capillary tubes and ow cytometry. to be higher compared to the same obtained Gel and solid phase methods offer some by using citrate and EDTA blood samples advantages over the tube method such as shows greater difference as indicated by the automation and technically easy to perform.9,10 Wilicoxon Signed Ranks Test Z and p-value as However, the essential principle remains the same. shown in Table 10. The difference is more so The indirect coomb’s test can be performed by with higher dilution samples which indicates serum or plasma while direct by red blood cells that there is a possibility that a low titre of of the blood samples. However, Lewis S.M. et al. anti-D IgG (both by direct antiglobulin test preferred serum to plasma for indirect coombs and indirect antiglobulin test) may be missed test, but also quoted that plasma is being used if EDTA blood samples are used compare to increasingly for convenience purpose.8 When plain blood samples. plasma is used, complement is inhibited by the The citrate blood samples gives intermediate EDTA anticoagulant, which effects the detection of pattern between plain blood samples and EDTA certain antibody (e.g. Kidd) or gives weak reaction blood samples. 8 with anti-D IgG. Baker et al. for direct coombs test Microscopy and grading of agglutination is mentioned that Red Blood Cells preferably from highly subjective. The present study is a rst of its clot should be used for performing antiglobulin kind. Extensive search in archives and internet has 11,12 test. The reagent to be used to modulate not revealed any such studies. This is the reason antibody binding in coombs test can be better why very minimal references are available. Understood by studying anti-Rh(D) antibodies on a molecular level.13 In the present work it is noticed that the degree of clumping in antiglobulin test Conclusion done with various dilutions of anti-D IgG is more with plain blood samples compared with those to From the results obtained and analyzed in the EDTA blood samples. An attempt is also made to present study the following conclusions are made:- compare the results of plain blood samples and 1. The use of plain blood is found to give better EDTA blood samples with Citrate blood samples. It results compared to citrate and EDTA blood is noticed that the result with citrate blood samples in performing the antiglobulin test. are in between those obtained from plain blood and EDTA blood. The above nding are consistent 2. The chances of missing the detection of low with the view of both Lewis et al. and Baker et al., titres of anti-D IgG is more with EDTA blood that plain blood is preferred in performing the samples when compared to citrate blood antiglobulin test. samples and least with plain blood samples. In the blood bank the donor’s blood is collected 3. Due to these reasons it is recommended to in ACD/CPD bags. After disconnecting the needle carry forward and have further studies and from the donors vein the blood in the tubing is made evolve a consensus on the use of plain or

Indian Journal of Pathology: Research and Practice / Volume 9 Number 1 (Part II)/January - April 2020 Siddhartha Shanker Sinha, Yogesh Kumar Yadav, Sonal Saxena, et al. 247

anticoagulated blood samples in reporting 6. Merry AH, Thomson EE, Rawlinson VI, et al. Coombs test. Quantitation of IgG on erythrocytes: correlation of number of IgG molecules per cell with the strength Acknowledgement: none of the direct and indirect antiglobulin tests. Vox Sang 1984;47:73–81. Conflict of Interest: none 7. Garratty G. Immune hemolytic anemia associated with negative routine serology. Semin Hematol 2005;42:156–64. Key Messages: For performing coombs test, use of plain blood instead of anticoagulated blood 8. Bain BJ, Bates I, Laffan MA, et al. Dacie and Lewis th gives better results. There is better probability Practical Haematology, 11 ed. Edinburgh: Elsevier Churchill Livingstone 2012.pp.229–44. detection of low titres of anti-D IgG with plain blood samples as compared to anticoagulated 9. Alwar V, Shantahla Devi AM, Sitalakshmi S, et blood (Ethylenediaminetetraacetic acid (EDTA) or al. Evaluation of the use of gel card system for assessment of direct Coombs test: Weighing the Citrate). pros and cons. Indian J Hematol Blood Transfus 2012;28(1):15–8. References 10. Moulds JM, Diekman L, Wells TD. Evaluation of column technology for direct antiglobulin testing. Immunohematology 1998;14(4):146–8. 1. Coombs RR, Mourant AE, Race RR. A new test for the detection of weak and incomplete Rh 11. F. J. Baker. Blood group technique. In: Introduction th agglutinins. Br J Exp Pathol 1945;26:255–66. to medical laboratory technology 5 ed F. J. Baker and R.E. Silverton, London: The English Language 2. Rosse WF. The antiglobulin test in autoimmune Book Society and Butterworths publishers 1978. hemolytic anemia. Annu Rev Med 1975;26:331–6. pp.673–4. 3. Rumsey DH, Ciesielski DJ. New protocols in 12. Linda A. Smith. Hemolytic Anemia: Immune serologic testing: A review of techniques to anemias. In: Clinical laboratory hematology, 2nd ed. meet today’s challenges. Immunohematology Shirlyn B. Mckenzie and J. Lynne Williams, New 2000;16:131–7. Jersey: Pearson clinical laboratory science series 4. Widmann FK, Barnes A, Case J, et al. Technical 2010.pp.345–7. th Manual, 8 ed. Washington DC: AABB; 1981:422– 13. Chang T Y, Siegel D L. Genetic and immunological 516. properties of phage-displayed human anti-Rh(D) 5. McGann PT, McDade J, Mortier NA, et al. IgA- antibodies: Implications for Rh(D) epitope topology. mediated autoimmune hemolytic anemia in an Amer J Hematol 1998;91:3066–78. infant. Pediatr Blood Cancer 2011;56:837–9.

Indian Journal of Pathology: Research and Practice / Volume 9 Number 1 (Part II)/January - April 2020